Introduction: Insulin and C-peptide assessment are important in characterization and management of diabetes. However, their adoption and increased clinical use in low resource settings (LRSs) is partly hindered by logistical factors including supplies required for pre-analytical sample handling and limited infrastructure. We aimed to determine the effects of altered sample processing conditions on stability of insulin and C-peptide at the pre-analytical stage. Methods: We investigated the stability of C-peptide and insulin in serum and plasma collected, preservative type, time to centrifugation, storage conditions and duration of storage on the stability of C-peptide and insulin over 24 hours. Results: Both C-peptide and insulin levels remained stable above 90% from baseline p=1.000 & p=0.776 over 24 hours for samples stored in K2EDTA tubes, whether at room temperature or in a cooler box, both as centrifuged and uncentrifuged whole blood. In contrast, samples collected in plain serum tubes kept at room temperature and uncentrifuged C-peptide and insulin levels decreased significantly to 51%, p=0.006 and 62%, p=0. 083 respectively, similarly insulin levels for centrifuged samples declined to 64%, p=0.083 All iced and centrifuged serum samples remained above 90% of baseline concentration. Conclusions: In resource limited settings where insulin and c-peptide tests are limited to central laboratories and highly dependent on sample referral systems, these tests can be reliably measured without the need for immediate centrifugation or processing from samples collected in whole blood K2EDTA tubes uncentrifuged kept at room temperature and processed within 24hours.